Chiaki Kajiwara

Heat shock protein 90 (HSP90) contributes to cytosolic translocation of extracellular antigen for cross-presentation by dendritic cells

In antigen (Ag) cross-presentation, dendritic cells (DCs) take up extracellular Ag and translocate them from the endosome to the cytosol for proteasomal degradation. The processed peptides can enter the conventional MHC I pathway. The molecules responsible for the translocation of Ag across the endosomal membrane into the cytosol are unknown. Here we demonstrate that heat shock protein 90 (HSP90) is critical for this step. Cross-presentation and -priming were decreased in both HSP90α-null DCs and mice. CD8α+ DC apoptosis mediated by translocation of exogenous cytochrome c to the cytosol was also eliminated in HSP90α-null mice. Ag translocation into the cytosol was diminished in HSP90α-null DCs and in DCs treated with an HSP90 inhibitor. Internalized Ag was associated with HSP90 and translocated to the cytosol, a process abrogated by the HSP90 inhibitor. Ag within purified phagosomes was released in an HSP90-dependent manner. These results demonstrate the important role of HSP90 in cross-presentation by pulling endosomal Ag out into the cytosol.

Essential Role of Endogenous Heat Shock Protein 90 of Dendritic Cells in Antigen Cross-Presentation

Extracellular HSP90 associated with Ag peptides have been demonstrated to efficiently cross-prime T cells, following internalization by dendritic cells (DCs). In addition, the nature of cell-associated Ags required for cross-priming is implicated as peptides and proteins chaperoned by heat shock protein (HSP). However, the role of endogenous HSP in DCs during cross-presentation remains elusive. In this paper, we show that endogenous HSP90 is essential for cross-presentation of both soluble and cell-associated Ags in DCs. Cross-presentation of soluble OVA and OVA-loaded transporter associated with Ag processing-1–deficient cells by bone marrow-derived DCs and DC-like cell line DC2.4 was profoundly blocked by HSP90 inhibitors, whereas presentation of endogenously expressed OVA was only partially suppressed. Assays using small interfering RNA and heat shock factor-1–deficient DCs (with defective expression of HSP90α) revealed the pivotal role of HSP90α in cross-presentation. The results suggest that in addition to HSP90 in Ag donor cells, endogenous HSP90 in DCs plays an essential role during Ag cross-presentation and, moreover, points to a link between heat shock factor-1–dependent induction of HSP90α within DC and cytotoxic T cell immunity.

Both CD4+ and CD8+ T cell epitopes fused to heat shock cognate protein 70 (hsc70) can function to eradicate tumors

Vaccination with heat shock proteins (HSP) protects mice from challenge with the tumor from which the HSP were isolated. The antigenicity of HSP vaccination is thought to result from HSP‐associated endogenous major histocompatibility complex class I peptides or their precursors. The vaccination effect can be achieved in an adjuvant‐free manner and is mediated by CD8+ T cells, indicating that HSP can act as a natural adjuvant and cross‐prime T cells in vivo. We previously devised a recombinant vaccine composed of a CD8+ T cell epitope fused to the carboxyl‐terminus of hsc70 and demonstrated efficient generation of antigen‐specific cytotoxic T lymphocyte (CTL) after vaccination with a few micrograms of the hsc70‐CTL epitope fusion protein. The present study aimed to determine if the fusion protein vaccine could control tumor growth in vivo and whether simultaneous fusion of a CD4+ T cell epitope to the amino terminus of the hsc70‐CTL epitope would be a more potent vaccine compared to the CTL epitope alone. Ovalbumin (OVA)–derived 8 mer peptide, OVA257‐264, and 16mer peptide, OVA265‐280, were used as CD8+ and CD4+ T cell epitopes, respectively. Vaccination with hsc70‐OVA257‐264 generated peptide specific CTL more effectively than a peptide plus incomplete Freunds adjuvant combination, and suppressed growth of OVA expressing EL4 (E.G7) and B16 melanoma tumor cells. Addition of OVA265‐280 to the amino‐terminus of hsc70‐OVA257‐264 (OVA265‐280‐hsc70‐OVA257‐264) enhanced the generation of the OVA257‐264‐specific CTL population, leading to better eradication of MO5 lung metastasis compared to hsc70‐OVA257‐264. Our results suggest that fusion of both CD4+ and CD8+ T cell epitopes to hsc70 enhances tumor immunity beyond the effect of the CD8+ T cell epitope alone. (Cancer Sci 2008; 99: 1008–1015)

HSP70 and HSP90 Differentially Regulate Translocation of Extracellular Antigen to the Cytosol for Cross-Presentation

Antigens (Ag) from cancer or virus-infected cells must be internalized by dendritic cells (DCs) to be presented to CD8+ T cells, which eventually differentiate into Ag-specific cytotoxic T lymphocytes (CTLs) that destroy cancer cells and infected cells. This pathway is termed cross-presentation and is also implicated as an essential step in triggering autoimmune diseases such as Type I diabetes. Internalized Ag locates within endosomes, followed by translocation through a putative pore structure spanning endosomal membranes into the cytosol, where it is degraded by the proteasome to generate antigen peptides. During translocation, Ag is believed to be unfolded since the pore size is too narrow to accept native Ag structure. Here, we show that paraformaldehyde-fixed, structurally inflexible Ag is less efficient in cross-presentation because of diminished translocation into the cytosol, supporting the “unfolded Ag” theory. We also show that HSP70 inhibitors block both endogenous and cross-presentation. ImageStream analysis revealed that the inhibition in cross-presentation is not due to blocking of Ag translocation because a HSP70 inhibitor rather facilitates the translocation, which is in marked contrast to the effect of an HSP90 inhibitor that blocks Ag translocation. Our results indicate that Ag translocation to the cytosol in cross-presentation is differentially regulated by HSP70 and HSP90.

Differential MyD88/IRAK4 requirements for cross-priming and tumor rejection induced by heat shock protein 70-model antigen fusion protein.

Priming of CD8+ T cells requires two signals, one produced by T‐cell receptor recognition of antigen, and a second that is often provided by the innate immune response. In this context, antigens non‐covalently or covalently associated with heat shock proteins (HSP) are internalized and processed in antigen‐presenting cells (APC) to be presented by MHC I molecules to CD8+ T cells, thus, signal 1 has been well characterized in this pathway of cross‐presentation. Signal 2 is not fully understood, although there are reports that Toll‐like receptors (TLRs) interact with HSP and activate APC. The ability of HSP to activate APC through TLRs is, however, controversial because of the possibility of endotoxin contamination. Using a variety of TLR KO mice, we present evidence that TLRs (TLR2, 3, 4, 7, and 9) and their adaptor molecules MyD88 and IRAK4 are dispensable in cross‐priming by a mycobacterial HSP70–antigen (ovalbumin as a model antigen) fusion protein; in contrast, MyD88/IRAK4, but not TLRs, are required for tumor rejection induced by the same reagent. Our results indicate that HSP‐mediated cross‐priming uses a second signal produced by mechanisms other than TLR cascades. We hypothesize that efficient cross‐priming by HSP70 alone is insufficient for tumor rejection and that MyD88/IRAK4‐dependent inflammatory stimulation, which might contribute to maintenance of the initially primed effector cells, is required to eradicate tumor burden. (Cancer Sci 2012; 103: 851–859)

Spermatogenesis arrest caused by conditional deletion of Hsp90α in adult mice

Summary It is controversial whether a functional androgen receptor (AR) on germ cells, including spermatogonia, is essential for their development into sperm and, thus, initiation and maintenance of spermatogenesis. It was recently shown that many spermatocytes underwent apoptosis in the testes of Hsp90&agr; KO mice. We had generated Hsp90&agr; KO mice independently and confirmed this phenotype. However, the important question of whether Hsp90&agr; is required to maintain spermatogenesis in adult mice in which testicular maturation is already completed could not be addressed using these conventional KO mice. To answer this question, we generated a tamoxifen-inducible deletion mutant of Hsp90&agr; and found that conditional deletion of Hsp90&agr; in adult mice caused even more severe apoptosis in germ cells beyond the pachytene stage, leading to complete arrest of spermatogenesis and testicular atrophy. Importantly, immunohistochemical analysis revealed that AR expression in WT testis was more evident in spermatogonia than in spermatocytes, whereas its expression was aberrant and ectopic in Hsp90&agr; KO testis, raising the possibility that an AR abnormality in primordial germ cells is involved in spermatogenesis arrest in the Hsp90&agr; KO mice. Our results suggest that the AR, specifically chaperoned by Hsp90&agr; in spermatogonia, is critical for maintenance of established spermatogenesis and for survival of spermatocytes in adult testis, in addition to setting the first wave of spermatogenesis before puberty.

Endogenous IL-17 as a factor determining the severity of Clostridium difficile infection in mice.

Clostridium difficile infection (CDI) is a toxin-mediated intestinal disease. Toxin A, toxin B and binary toxin are believed to be responsible for the pathogenesis of CDI, which is characterized by massive infiltration of neutrophils at the infected intestinal mucosa. IL-17 is one of the cytokines that play critical roles in several inflammatory and immunological diseases through various actions, including promoting neutrophil recruitment. The aim of this study was to examine the role of this cytokine in CDI by employing IL-17 A and F double knockout (IL-17 KO) mice for the CDI model. We demonstrated that IL-17 KO mice were more resistant to CDI than WT mice using several factors, such as diarrhoea score, weight change and survival rate. Although the bacterial numbers of C. difficile in faeces were not different, the inflammatory mediator levels at the large intestine on day 3 post-infection were attenuated in IL-17 KO mice. Finally, we showed that infiltration of neutrophils, but not macrophages, in the large intestine was significantly decreased in IL-17 KO mice compared to WT mice. In conclusion, the data demonstrate that endogenous IL-17 may be a factor determining the severity of CDI in mice. Although the mechanism is totally unknown, IL-17-mediated inflammatory responses, such as cytokine/chemokine production and neutrophil accumulation, may be plausible targets for future investigations.

Efficacy of β-Lactam-plus-Macrolide Combination Therapy in a Mouse Model of Lethal Pneumococcal Pneumonia

ABSTRACT Community-acquired pneumonia is a common disease with considerable morbidity and mortality, for which Streptococcus pneumoniae is accepted as a leading cause. Although β-lactam-plus-macrolide combination therapy for this disease is recommended in several guidelines, the clinical efficacy of this strategy against pneumococcal pneumonia remains controversial. In this study, we examined the effects of β-lactam-plus-macrolide combination therapy on lethal mouse pneumococcal pneumonia and explored the mechanisms of action in vitro and in vivo. We investigated survival, lung bacterial burden, and cellular host responses in bronchoalveolar lavage fluids obtained from mice infected with pneumonia and treated with ceftriaxone, azithromycin, or both in combination. Although in vitro synergy was not observed, significant survival benefits were demonstrated with combination treatment. Lung neutrophil influx was significantly lower in the ceftriaxone-plus-azithromycin-treated group than in the ceftriaxone-treated group, whereas no differences in the lung bacterial burden were observed on day 3 between the ceftriaxone-plus-azithromycin-treated group and the ceftriaxone-treated group. Notably, the analysis of cell surface markers in the ceftriaxone-plus-azithromycin combination group exhibited upregulation of presumed immune checkpoint ligand CD86 and major histocompatibility complex class II in neutrophils and CD11b-positive CD11c-positive (CD11b+ CD11c+) macrophages and dendritic cells, as well as downregulation of immune checkpoint receptors cytotoxic-T lymphocyte-associated antigen 4 and programmed death 1 in T helper and T regulatory cells. Our data demonstrate that the survival benefits of ceftriaxone-plus-azithromycin therapy occur through modulation of immune checkpoints in mouse pneumococcal pneumonia. In addition, immune checkpoint molecules may be a novel target class for future macrolide research.

HSP90α deficiency does not affect immunoglobulin gene hypermutation and class switch but causes enhanced MHC class II antigen presentation

Heat shock protein 90 (HSP90) is a molecular chaperone required for efficient antigen presentation and cross-presentation. In addition, HSP90 was recently reported to interact with and stabilize the activation-induced cytidine deaminase (AID) and plays a critical role in immunoglobulin gene hypermutation and class switch recombination. In mice and humans, there are two HSP90 isoforms, HSP90α and HSP90β, but the in vivo role of each isoform remains largely unknown. Here we have analyzed humoral immune responses in HSP90α-deficient mice. We found that HSP90α deficiency did not affect AID protein expression. B cell development and maturation, as well as immunoglobulin gene hypermuation and class switch, occurred normally in HSP90α-deficient mice. However, antibody production to a T-dependent antigen was elevated in the mutant mice and this was associated with enhanced MHC class II antigen presentation to T helper cells by dendritic cells. Our results reveal a previously unidentified inhibitory role for HSP90α isoform in MHC class II antigen presentation and the humoral immune response. Along with our recent finding that HSP90α is required for antigen cross-presentation, these results suggest that HSP90α controls the balance of humoral and cellular immunity by dictating the fate of presentation of exogenous antigen.

Extracellular and Non-Chaperone Function of Heat Shock Protein−90α Is Required for Skin Wound Healing

Despite years of effort and investment, there are few topical or systemic medications for skin wounds. Identifying natural drivers of wound healing could facilitate the development of new and effective treatments. When skin is injured, there is a massive increase of heat shock protein (Hsp) 90α inside the wound bed. The precise role for these Hsp90α proteins, however, was unclear. The availability of a unique mouse model that lacked the intracellular ATPase-driven chaperoning, but spared the extracellular fragment-5-supported pro-motility function of Hsp90α allowed us to test specifically the role of the non-chaperone function of Hsp90α in normal wound closure. We found that the chaperone-defective Hsp90α-Δ mutant mice showed similar wound closure rate as the wild-type Hsp90α mice. We generated recombinant proteins from the mouse cDNAs encoding the Hsp90α-Δ and wild-type Hsp90α. Topical application of Hsp90α-Δ mutant protein promoted wound closure as effectively as the full-length wild-type Hsp90α protein. More importantly, selective inhibition of the extracellular Hsp90α-Δ protein function by a monoclonal antibody targeting the fragment-5 region disrupted normal wound closure in both wild-type Hsp90α and Hsp90α-Δ mice. Thus, this study provides direct support for non-chaperone, extracellular Hsp90α as a potential driver for normal wound closure.